CNG2B: a novel human cyclic nucleotide-gated ion channel

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of CNG2B, antibodies to CNG2B, methods of detecting CNG2B, and methods of screening for modulators of cyclic nucleotide-gated ion channels using biologically active CNG2B. The invention further provides, in a computer system, a method of screening for mutations of human CNG2B genes as well as a method for identifying a three-dimensional structure of human CNG2B polypeptides.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Ser. No. 60/226,253,filed Aug. 17, 2000, herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofCNG2B, antibodies to CNG2B, methods of detecting CNG2B, and methods ofscreening for modulators of cyclic nucleotide-gated cation channelsusing biologically active CNG2B. The invention further provides, in acomputer system, a method of screening for mutations of human CNG2Bgenes as well as a method for identifying a three-dimensional structureof human CNG2B polypeptides.

BACKGROUND OF THE INVENTION

Cyclic nucleotide gated cation channels (CNG) are a class ofnon-selective cation channels that are opened by direct binding ofcyclic nucleotides such as cGMP and cAMP. CNG channels are highlypermeable to Na⁺ and Ca²⁺ and their activation leads to depolarizationand increases in internal Ca²⁺ concentrations. These channels can linkchanges in cytoplasmic cyclic nucleotide levels to changes in cellularexcitability, secretion of neurotransmitters and the stimulation ofcalcium-dependent pathways.

CNG family channel proteins are multimers and can be formed by at leasttwo functionally distinct classes of subunits. The two classes ofsubunits, alpha and beta, share a common motif of 6 transmembranedomains, a pore motif and a cytoplasmic cyclic nucleotide binding domain(Finn et al., Annu. Rev. Physiol. 58:395-426:1996). CNG alpha subunitscan form functional channels as homomultimers, i.e., all subunitscontributing to the channel pore are identical. Beta subunits, incontrast, can only form functional channels when expressed with an alphasubunit. These heteromultimeric channels show functional propertiesconsistent with native CNG channels (Gerstner, et al., J. Neurosci. 20(4):1324-1332, 2000; Finn, et al., Annu. Rev. Physiol. 58:395-426,1996). For example, coexpression of alpha and beta subunits occurs inretinal rod cells where the alpha subunit CNGA1 forms a heteromultimerwith the beta subunit CNGB1 (CNG4) (Gerstner, et al., J. Neurosci. 20(4):1324-1332, Feb. 15, 2000).

CNG channels are important for sensory signal transduction in retinaland olfactory and taste bud cells in response to primary sensory stimulisuch as light and aerosolized or dissolved molecules (Ding, C, et al.,Am. J. Physiol. 272 (Cell Physiol. 41): C1335-C1344, 1997). Inphotoreceptor cells, CNG channels are open in darkness due to a highbasal concentration of cGMP. This causes a tonic depolarization of themembrane and constitutive neurotransmitter release. Upon stimulation bylight, cGMP levels drop, closing the CNG channels. This in turn causes ahyperpolarization of the membrane, a drop in the internal Ca²⁺concentration, and a decrease in the release of neurotransmitter (Finn,et al., Annu. Rev. Physiol. 58:395-426, 1996).

CNG channels have been found in a number of tissues, suggesting thatthese channels may link a variety of stimuli to changes in membranepotential and cytoplasmic calcium levels (Ding, et al., Am. J. Physiol.272 (Cell Physiol. 41):C1335-C1344, 1997; Kingston P, Synapse 32:1-12,1999). For instance, retinal and olfactory CNG channels are expressed invarious parts of the brain (Ding, et al., Am. J. Physiol. 272 (CellPhysiol. 41):C1335-C1344, 1997; Kingston P, Synapse 32:1-12, 1999).Because these channels are highly permeable to Ca²⁺, they may stimulateCa²⁺-dependent pathways that have significant effects on neuronalactivity. More directly, they may contribute to neuronal activity byproviding excitatory depolarizations. CNG channels may also interactwith other second messenger systems such as the Nitric Oxide-pathway toprovide the longer lasting changes that are important mechanisms inlearning and memory (Kingston, Synapse 32:1-12, 1999). CNG channels havebeen found in the testis, and through the regulation of the internalCa²⁺ concentration, may be involved in chemotaxis of sperm (Weyand, etal., Nature 368:859-863, 1994). Expression of CNG channels has also beennoted in heart, aorta and kidney, where they may play a role in theregulation of heart rate, blood pressure and electrolyte transport,respectively (Finn et al., Ann. Rev. Physiol. 1996, 58:395-426). Thefill scope of CNG channel function is not yet entirely understood, butit is clear that they play a key role in many physiological processes.

SUMMARY OF THE INVENTION

The current invention provides the first isolation and characterizationof human CNG2B, a novel subunit of a cyclic nucleotide gated cationchannel. The present invention provides both the nucleotide and aminoacid sequence of CNG2B, as well as methods of assaying for modulators ofCNG2B, antibodies to CNG2B, and methods of detecting CNG2B nucleic acidsand proteins.

In one aspect, the present invention provides an isolated nucleic acidencoding a polypeptide comprising a subunit of a cation channel, thepolypeptide: (i) forming, with at least one CNG alpha subunit, a cationchannel having the characteristic of cyclic nucleotide-gating or nitricoxide gating; and (ii) comprising an amino acid sequence having at least95% identity to SEQ ID NO:1.

In another aspect, the present invention provides an isolated nucleicacid encoding a CNG2B polypeptide, the nucleic acid specificallyhybridizing under stringent conditions to a nucleic acid comprising anucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3.

In another aspect, the present invention provides an isolated nucleicacid encoding a CNG2B polypeptide, the nucleic acid comprising anucleotide sequence having at least 90% sequence identity to SEQ ID NO:2or SEQ ID NO:3.

In one embodiment, the nucleic acid encodes a polypeptide comprising anamino acid sequence of SEQ ID NO:1. In another embodiment, the nucleicacid comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3.

In another embodiment, the nucleic acid is amplified by primers thatselectively hybridize under stringent hybridization conditions to thesame sequence as the primers selected from the group consisting of:GCAGATCTTTCAGAACTGTGAGGCCA (SEQ ID NO:4) CCTGCCCTCTTCATCTTTGGAAGTTC (SEQID NO:5) GCCAACATCAAGAGCCTAGGTTATTC (SEQ ID NO:6)GGATGATCTACAGACCAAGTTTGCTCG (SEQ ID NO:7) ATGAGCCAGGACACCAAAGTGAAGAC(SEQ ID NO:8) GTTGATGATGCTGATCTCCCCAAAG (SEQ ID NO:9)GGATGATGAGGTTATACATGACTGGG (SEQ ID NO:10) AGGCTAGCAACTTCCTGGCCTTGGAT(SEQ ID NO:11) GCGAAAGCTTCCACCATGAGCCAGGACACCAAAGTG (SEQ ID NO:12) andCATGTCTAGAATGGGGATGGGGTCACTCTGGACCT. (SEQ ID NO:13)

In another embodiment, the nucleic acid selectively hybridizes undermoderately stringent hybridization conditions to a nucleic acidcomprising a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3. Inanother aspect, the present invention provides an isolated nucleic acidthat specifically hybridizes under stringent conditions to a nucleicacid encoding an amino acid sequence of SEQ ID NO:1.

In another aspect, the present invention provides a method of detectinga nucleic acid, the method comprising contacting the nucleic acid withan isolated nucleic acid, as described above.

In another aspect, the present invention provides expression vectorscomprising the nucleic acids of the invention, and host cells comprisingsuch expression vectors.

In another aspect, the present invention provides an isolatedpolypeptide comprising a subunit of a cation channel, the polypeptide:(i) forming, with at least one CNG alpha subunit, a cation channelhaving the characteristic of cyclic nucleotide-gating; and (ii)comprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO:1.

In one embodiment, the polypeptide specifically binds to antibodiesgenerated against a polypeptide comprising an amino acid sequence of SEQID NO:1. In another embodiment, the polypeptide comprises an alphasubunit of a homomeric cyclic nucleotide gated cation channel. Inanother embodiment, the polypeptide comprises an alpha subunit of aheteromeric cyclic nucleotide gated cation channel. In anotherembodiment, the polypeptide has a molecular weight of between about 61kD to about 71 kD. In another embodiment, the polypeptide has an aminoacid sequence of human CNG2B. In another embodiment, the polypeptide hasan amino acid sequence of SEQ ID NO:1.

In another aspect, the present invention provides an antibody thatspecifically binds to any of the CNG2B polypeptides described herein.

In another aspect, the present invention provides a method foridentifying a compound that increases or decreases ion flux through acation channel, the method comprising the steps of: (i) contacting thecompound with a CNG2B polypeptide, the polypeptide (a) forming, with atleast one CNG alpha subunit, a cation channel having the characteristicof cyclic nucleotide-gating; and (b) comprising an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:1; and (ii)determining the functional effect of the compound upon the cationchannel.

In one embodiment, the functional effect is a physical effect or achemical effect. In one embodiment, the polypeptide is recombinant. Inanother embodiment, the functional effect is determined by measuringligand binding to the channel. In another embodiment, the cation channelis homomultimeric. In another embodiment, the cation channel isheteromultimeric.

In one embodiment, the polypeptide is expressed in a eukaryotic hostcell or cell membrane. In another embodiment, the functional effect isdetermined by measuring ion flux, changes in ion concentrations, changesin current or changes in voltage.

In another aspect, the present invention provides a method foridentifying a compound that increases or decreases ion flux through acyclic nucleotide-gated cation channel comprising a CNG2B polypeptide,the method comprising the steps of: (i) entering into a computer systeman amino acid sequence of at least 100 amino acids of a CNG2Bpolypeptide or at least 300 nucleotides of a nucleic acid encoding theCNG2B polypeptide, the CNG2B polypeptide comprising an amino acidsequence having at least 95% sequence identity to SEQ ID NO:1; (ii)generating a three-dimensional structure of the polypeptide encoded bythe amino acid sequence; (iii) generating a three-dimensional structureof the cation channel comprising the CNG2B polypeptide; (iv) generatinga three-dimensional structure of the compound; and (v) comparing thethree-dimensional structures of the polypeptide and the compound todetermine whether or not the compound binds to the polypeptide.

In one embodiment, the amino acid sequence is of a full-length CNG2Bpolypeptide.

In another aspect, the present invention provides a method of modulatingion flux through a CNG cation channel comprising a CNG2B subunit totreat a disease in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of acompound identified using any of the methods described herein.

In another aspect, the present invention provides a method of detectingthe presence of CNG2B in human tissue, the method comprising the stepsof: (i) isolating a biological sample; (ii) contacting the biologicalsample with an CNG2B-specific reagent that selectively associates withCNG2B; and, (iii) detecting the level of CNG2B-specific reagent thatselectively associates with the sample.

In one embodiment, the CNG2B-specific reagent is selected from the groupconsisting of: CNG2B-specific antibodies, CNG2B-specific oligonucleotideprimers, and CNG2B-nucleic acid probes.

In another aspect, the present invention provides, in a computer system,a method of screening for mutations of a human CNG2B gene, the methodcomprising the steps of: (i) entering into the computer a first nucleicacid sequence encoding a CNG2B polypeptide having a nucleotide sequenceof, SEQ ID NO:2 or SEQ ID NO:3, and conservatively modified versionsthereof; (ii) comparing the first nucleic acid sequence with a secondnucleic acid sequence having substantial identity to the first nucleicacid sequence; and (iii) identifying nucleotide differences between thefirst and second nucleic acid sequences.

In one embodiment, the second nucleic acid sequence is associated with adisease state.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid alignment of CNG2B with rat OCNC2. Identical residuesare shaded and numbers at the left margin indicate amino acid position.

FIG. 2. Complete CNG2B sequence derived from assembly of PCR fragments.Coding sequence is in bold type, and untranslated sequence is in normaltype.

FIG. 3. Complete CNG2B coding nucleotide sequence.

FIG. 4. Complete CNG2B amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides for the first time nucleic acids encodingCNG2B, a member of the CNG family of cyclic nucleotide gated cationchannels. Members of this family are polypeptide subunits of cationchannels having six transmembrane regions, a pore motif, and acytoplasmic cyclic nucleotide binding domain. CNG2B is most similar torat OCNC2 which, without being bound to any particular theory, hascharacteristics of both alpha and beta subunits. Because CNG2B isexpressed in the central nervous system, modulators of CNG2B functioncan be identified which would be useful in the treatment of any of alarge number of neurological disorders.

The invention therefore provides methods of screening for activators andinhibitors of cation channels that contain a CNG2B subunit. Suchmodulators of cation channel activity are useful for treating disorders,including neurological disorders.

Furthermore, the invention provides assays for CNG activity where CNG2Bacts as a direct or indirect reporter molecule. Such uses of CNG2B as areporter molecule in assay and detection systems have broadapplications, e.g., CNG2B can be used as a reporter molecule to measurechanges in cation concentration, membrane potential, current flow, ionflux, transcription, signal transduction, receptor-ligand interactions,second messenger concentrations, in vitro, in vivo, and ex vivo. In oneembodiment, CNG2B can be used as an indicator of current flow in aparticular direction (e.g., outward or inward cation flow), and inanother embodiment, CNG2B can be used as an indirect reporter viaattachment to a second reporter molecule such as green fluorescentprotein.

The invention also provides for methods of detecting CNG2B nucleic acidand protein expression, allowing investigation of the channel diversityprovided by CNG2B family members, as well as diagnosis of disorders,including neurological disorders.

Finally, the invention provides for a method of screening for mutationsof CNG2B genes or proteins. The invention includes, but is not limitedto, methods of screening for mutations in CNG2B with the use of acomputer. Similarly, the invention provides for methods of identifyingthe three-dimensional structure of CNG2B polypeptides, as well as theresulting computer readable images or data that comprise the threedimensional structure of CNG2B polypeptides. Other methods for screeningfor mutations of CNG2B genes or proteins include high densityoligonucleotide arrays, PCR, immunoassays and the like.

Functionally, CNG2B polypeptides are subunits, e.g., alpha subunits, ofcyclic nucleotide-gated cation channels. CNG2B-containing channels areeither homomultimeric or heteromultimeric. HeteromultimericCNG2B-containing channels can contain, in addition to the CNG2Bsubunits, one or more CNG alpha or beta subunits. The presence of CNG2Bin a cation channel may modulate the activity of the heteromeric channeland thus enhance channel diversity. Channel diversity is also enhancedwith alternatively spliced forms of CNG2B genes. CNG2B nucleic acidshave been isolated from cDNAs from the human central nervous system.

Structurally, the nucleotide sequence of human CNG2B (SEQ ID NOS:2-3)encodes a polypeptide monomer with a predicted molecular weight ofapproximately 66 kD and a predicted molecular weight range of 61-71 kD.CNG2B polypeptides typically contain each of the motifs common amongalpha and beta CNG subunits, including six transmembrane domains, a poremotif, and a cytoplasmic cyclic nucleotide binding domain (Finn et al.,Ann. Rev. Physiol. 58:395-426:1996). Related CNG2B genes from otherspecies share at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or,preferably, 95% to 100% amino acid identity with the CNG2B shown as SEQID NO:1.

The present invention also provides polymorphic variants of the humanCNG2B depicted in SEQ ID NO:1: variant #1, in which an isoleucineresidue is substituted for the valine residue at amino acid position110; variant #2, in which a glycine residue is substituted for theserine residue at amino acid position 520; variant #3, in which a lysineresidue is substituted for the arginine residue at amino acid position537; and variant #4, in which a glutamic acid residue is substituted forthe aspartic acid residue at amino acid position 550.

The CNG2B nucleotide and amino acid sequence may be used to identifyCNG2B polymorphic variants, interspecies homologs, and alleles. Thisidentification can be made in vitro, e.g., under stringent hybridizationconditions and sequencing, or by using the sequence information in acomputer system for comparison with other nucleotide sequences, or usingantibodies raised against CNG2B. Typically, identification of CNG2Bpolymorphic variants, orthologs, and alleles is made by comparing theamino acid sequence (or the nucleic acid encoding the amino acidsequence) of SEQ ID NO:1. Amino acid identity of approximately at least60% or above, 70%, 65%, 75%, 80%, preferably 85%, most preferably 95%,96%, 97%, 98%, or 99% typically demonstrates that a protein is a CNG2Bpolymorphic variant, interspecies homolog, or allele. Sequencecomparison is typically performed using the BLAST or BLAST 2.0 algorithmwith default parameters, discussed below.

CNG2B polymorphic variants, interspecies homologs, and alleles can beconfirmed by expressing or co-expressing the putative CNG2B polypeptidemonomer and examining whether it forms a cation channel with CNG familyfunctional and biochemical characteristics. This assay is used todemonstrate that a protein having about 60% or greater, 65%, 70%, 75%,80%, preferably 85%, 90%, or 95% or greater amino acid identity to CNG2Bshares the same functional characteristics as CNG2B and is therefore aspecies of CNG2B. Typically, human CNG2B having the amino acid sequenceof SEQ ID NO:1 is used as a positive control in comparison to theputative CNG2B protein to demonstrate the identification of a CNG2Bpolymorphic variant, ortholog, conservatively-modified variant, mutant,or allele.

CNG2B nucleotide and amino acid sequence information may also be used toconstruct models of cyclic nucleotide-gated cation channels in acomputer system. These models are subsequently used to identifycompounds that can activate or inhibit cyclic nucleotide-gated cationchannels comprising CNG2B polypeptides. Such compounds that modulate theactivity of channels comprising CNG2B polypeptides can be used toinvestigate the role of CNG2B polypeptides in the modulation of channelactivity and in channel diversity.

The isolation of biologically active CNG2B for the first time provides ameans for assaying for inhibitors and activators of cyclicnucleotide-gated cation channels that comprise CNG2B subunits.Biologically active CNG2B polypeptides are useful for testing inhibitorsand activators of cyclic nucleotide-gated cation channels comprisingsubunits of CNG2B, using in vivo and in vitro expression that measure,e.g., changes in voltage or current. Such activators and inhibitorsidentified using a cation channel comprising at least one CNG2B subunit,optionally up to four CNG2B subunits, can be used to further studycyclic nucleotide-gating, channel kinetics and conductance properties ofcation channels. Such activators and inhibitors are useful aspharmaceutical agents for treating diseases involving abnormal ion flux,e.g., disorders, including neurological disorders, as described above.Methods of detecting CNG2B nucleic acids and polypeptides and expressionof channels comprising CNG2B polypeptides are also useful for diagnosticapplications for diseases involving abnormal ion flux, e.g., asdescribed above. For example, chromosome localization of the geneencoding human CNG2B can be used to identify diseases caused by andassociated with CNG2B. Methods of detecting CNG2B are also useful forexamining the role of CNG2B in channel diversity and modulation ofchannel activity.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise. “CNG2B” refers to a polypeptide that is asubunit or monomer of a cyclic nucleotide gated cation channel, and amember of the CNG family. When CNG2B is part of a cation channel, e.g.,a homomultimeric or heteromultimeric cation channel, the channel has thecharacteristic of cyclic nucleotide gating or nitric oxide gating. Theterm CNG2B therefore refers to CNG2B polymorphic variants, alleles,mutants, and interspecies homologs that: (1) have an amino acidsubsequence that has greater than about 60% amino acid sequenceidentity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 95%, 96%, 97%, 98% or99% or greater amino acid sequence identity, to a CNG2B sequence of SEQID NO:1; (2) bind to antibodies, e.g., polyclonal antibodies, raisedagainst an immunogen comprising an amino acid sequence of SEQ ID NO:1 ora fragment or conservatively modified variants thereof; (3) specificallyhybridize under stringent hybridization conditions to a sequence of SEQID NOS:2-3 and fragments and conservatively modified variants thereof;(4) have a nucleic acid subsequence that has greater than about 90%,preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotidesequence identity to SEQ ID NO:2 or SEQ ID NO:3; or (5) are amplified byprimers that specifically hybridize under stringent hybridizationconditions to the same sequence as a primer set selected from the groupconsisting of SEQ ID NOS:4-13.

The phrase “cyclic nucleotide-gated” activity or “cyclicnucleotide-gating” refers to a characteristic of a cation channelcomposed of individual polypeptide monomers or subunits. Generally,cyclic-nucleotide-gated cation channels are a class of non-selectivecation channels that are opened by direct binding of cyclic nucleotidessuch as cGMP and cAMP. CNG channels are highly permeable to Na⁺ andCa²⁺, and their activation leads to depolarization and increases ininternal Ca²⁺ concentrations. CNG channels can thus link changes incytoplasmic cyclic nucleotide levels to changes in cellularexcitability, secretion of neurotransmitters, and/or stimulation ofcalcium-dependent pathways. CNG channels play an important role insensory signal transduction in numerous cells, e.g., cells throughoutthe central nervous system, in response to primary sensory stimuli suchas light and aerosolized or dissolved molecules. In photoreceptor cells,CNG channels are open in darkness due to a high basal concentration ofcGMP, causing a tonic depolarization of the membrane and constitutiveneurotransmitter release. Upon stimulation by light, cGMP levels drop,closing the CNG channels, and in turn causing a hyperpolarization of themembrane, a drop in the internal Ca²⁺ concentration, and a decrease inneurotransmitter release. CNG channels may also interact with secondmessenger systems such as the nitric oxide pathway. In some cases, NOmay substitute for cyclic nucleotides in gating these channels (see,e.g., Broillet, et al., Neuron 18:951-958 (1997)).

“Homomeric channel” or “homomultimeric channel” refers to a CNG channelcomposed of identical alpha subunits, whereas “heteromeric channel” or“heteromultimeric channel” refers to a CNG channel composed of at leastone CNG alpha subunit, e.g., CNG2B, plus at least one other type ofalpha or beta subunit.

An “alpha subunit” is a polypeptide monomer that is an essential subunitof a CNG cation channel, as at least one alpha subunit is required tocreate a functional channel. Alpha subunits can form a homomultimericcationic channel, or can form a heteromultimeric channel comprisingother beta subunits or other heterologous alpha subunits. Any particularalpha subunit may participate in a variety of channel types in anorganism or in a cell, e.g., forming homomultimeric channels in one celltype, forming a heteromultimeric channel with a beta subunit in a secondcell type, and forming a third heteromultimeric channel with aheterologous alpha subunit in a third cell type.

A “beta subunit” is a polypeptide monomer that is an auxiliary subunitof a CNG cation channel composed of alpha subunits; however, betasubunits alone cannot form a channel (see, e.g., U.S. Pat. No.5,776,734). Beta subunits are known, for example, to increase the numberof channels by helping the alpha subunits reach the cell surface, changeactivation kinetics, and change the sensitivity of natural ligandsbinding to the channels. Beta subunits can be outside of the pore regionand associated with alpha subunits comprising the pore region. They canalso contribute to the external mouth of the pore region.

The phrase “functional effects” in the context of assays for testingcompounds affecting a channel comprising a CNG2B polypeptide includesthe determination of any parameter that is indirectly or directly underthe influence of the channel. It includes e.g., direct, physicaleffects, such as ligand binding, and indirect, chemical or phenotypiceffects, e.g., changes in ion flux and membrane potential, and otherphysiologic effects such as increases or decreases of transcription orhormone release. “Functional effects” include in vitro (biochemical orligand binding assays using, e.g., isolated protein, cell lysates orcell membranes), in vivo (cell- and animal-based assays), and ex vivoactivities.

“Determining the functional effect” refers to examining the effect of acompound that has a direct physical effect on a CNG2B subunit or channelcomprising a CNG2B subunit, e.g., ligand binding, or indirect chemicalor phenotypic effects on channel comprising a CNG2B subunit, e.g.,increases or decreases ion flux in a cell or cell membrane. The ion fluxcan be any ion that passes through a channel and analogues thereof,e.g., potassium, rubidium. Preferably, the term refers to the functionaleffect of the compound on the channels comprising CNG2B, e.g., changesin ion flux including radioisotopes, current amplitude, membranepotential, current flow, conductance, transcription, protein binding,phosphorylation, dephosphorylation, second messenger concentrations(cAMP, cGMP, Ca²⁺, IP₃), ligand binding, changes in ion concentration,and other physiological effects such as hormone and neurotransmitterrelease, as well as changes in voltage and current. Such functionaleffects can be measured by any means known to those skilled in the art,e.g., patch clamping, voltage-sensitive dyes, ion sensitive dyes, wholecell currents, radioisotope efflux, inducible markers, and the like.

“Inhibitors,” “activators” or “modulators” of cyclic nucleotide-gatedcation channels comprising a CNG2B polypeptide refer to inhibitory oractivating molecules identified using in vitro and in vivo assays forCNG2B channel function. Inhibitors are compounds that decrease, block,prevent, delay activation, inactivate, desensitize, or down regulate thechannel. Activators are compounds that increase, open, activate,facilitate, enhance activation, sensitize or up regulate channelactivity. Such assays for inhibitors and activators include e.g.,expressing a CNG2B polypeptide in cells or cell membranes and thenmeasuring flux of ions through the channel and determining changes inpolarization or Ca⁺ concentration (i.e., electrical potential).Alternatively, cells expressing endogenous CNG2B channels can be used insuch assays. To examine the extent of inhibition, samples or assayscomprising a CNG2B channel are treated with a potential activator orinhibitor and are compared to control samples without the inhibitor.Control samples (untreated with inhibitors) are assigned a relativeCNG2B activity value of 100%. Inhibition of channels comprising CNG2B isachieved when the CNG2B activity value relative to the control is about90%, preferably 50%, more preferably 25-0%. Activation of channelscomprising CNG2B is achieved when the CNG2B activity value relative tothe control is 110%, more preferably 150%, most preferably at least200-500% higher or 1000% or higher.

“Biologically active” CNG2B polypeptides refers to CNG2B polypeptidesthat have the ability to form a cation channel having the characteristicof cyclic nucleotide-gating tested as described herein.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. Inparticular, an isolated CNG2B nucleic acid is separated from openreading frames that flank the CNG2B gene and encode proteins other thanCNG2B. The term “purified” denotes that a nucleic acid or protein givesrise to essentially one band in an electrophoretic gel. Particularly, itmeans that the nucleic acid or protein is at least 85% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid (e.g., asphingolipid), fatty acid, polynucleotide, oligonucleotide, etc., to betested for the capacity to directly or indirectly modulation lymphocyteactivation. The test compound can be in the form of a library of testcompounds, such as a combinatorial or randomized library that provides asufficient range of diversity. Test compounds are optionally linked to afusion partner, e.g., targeting compounds, rescue compounds,dimerization compounds, stabilizing compounds, addressable compounds,and other functional moieties. Conventionally, new chemical entitieswith useful properties are generated by identifying a test compound(called a “lead compound”) with some desirable property or activity,e.g., inhibiting activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds. Often,high throughput screening (HTS) methods are employed for such ananalysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 5000 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins    (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 15 to 350 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins for which antisera or monoclonal antibodies are available(e.g., the polypeptide of SEQ ID NO:1 can be made detectable, e.g., byincorporating a radiolabel into the peptide, and used to detectantibodies specifically reactive with the peptide).

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

The term “heterolbgous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, 65%, 70%, 75%, 80%, preferably 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acidsequence such as SEQ ID NO:1 or a nucleotide sequence such as SEQ IDNO:2 or SEQ ID NO:3), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Preferably, the identity exists over aregion that is at least about 25 amino acids or nucleotides in length,or more preferably over a region that is 50-100 amino acids ornucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. For sequence comparison of nucleicacids and proteins to CNG2B nucleic acids and proteins, the BLAST andBLAST 2.0 algorithms and the default parameters discussed below areused.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff& Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments. (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993)). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole etal, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. (1985)). Techniques for the production of single chain antibodies(U.S. Pat. No. 4,946,778) can be adapted to produce antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

An “anti-CNG2B” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by a CNG2B gene, cDNA, or asubsequence thereof.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to CNG2B, as shown in SEQ ID NO:1, or splice variants, orportions thereof, can be selected to obtain only those polyclonalantibodies that are specifically immunoreactive with CNG2B and not withother proteins. This selection may be achieved by subtracting outantibodies that cross-react with molecules such as other CNG familymembers. In addition, polyclonal antibodies raised to CNG2B polymorphicvariants, alleles, orthologs, and conservatively modified variants canbe selected to obtain only those antibodies that recognize CNG2B, butnot other CNG family members. In addition, antibodies to human CNG2B butnot other CNG2B orthologs can be selected in the same manner. A varietyof immunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, e.g., cultured cells, explants, and cells in vivo.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains CNG2B polypeptides or nucleic acid encoding a CNG2Bprotein. Such samples include, but are not limited to, tissue isolatedfrom humans. Biological samples may also include sections of tissuessuch as frozen sections taken for histologic purposes. A biologicalsample is typically obtained from a eukaryotic organism, preferablyeukaryotes such as fungi, plants, insects, protozoa, birds, fish,reptiles, and preferably a mammal such as rat, mice, cow, dog, guineapig, or rabbit, and most preferably a primate such as chimpanzees orhumans.

III. Isolating a Gene Encoding a CNG2B Polypeptide

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (Kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, front sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kD) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingCNG2B Polypeptides

In general, the nucleic acid sequences encoding CNG2B and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries or isolated using amplification techniques witholigonucleotide primers. For example, CNG2B sequences are typicallyisolated from human nucleic acid (genomic or cDNA) libraries byhybridizing with a nucleic acid probe or polynucleotide, the sequence ofwhich can be derived from SEQ ID NOS:2-3. A suitable tissue from whichCNG2B RNA and cDNA can be isolated is the central nervous system (CNS).Preferably, the template for the amplification is first strand cDNA madefrom some part of the human CNS.

Amplification techniques using primers can also be used to amplify andisolate CNG2B from DNA or RNA. The following primers can also be used toamplify a sequence of human CNG2B: GCAGATCTTTCAGAACTGTGAGGCCA (SEQ IDNO:4) CCTGCCCTCTTCATCTTTGGAAGTTC (SEQ ID NO:5)GCCAACATCAAGAGCCTAGGTTATTC (SEQ ID NO:6) GGATGATCTACAGACCAAGTTTGCTCG(SEQ ID NO:7) ATGAGCCAGGACACCAAAGTGAAGAC (SEQ ID NO:8)GTTGATGATGCTGATCTCCCCAAAG (SEQ ID NO:9) GGATGATGAGGTTATACATGACTGGG (SEQID NO:10) AGGCTAGCAACTTCCTGGCCTTGGAT (SEQ ID NO:11)GCGAAAGCTTCCACCATGAGCCAGGACACCAAAGTG (SEQ ID NO:12) andCATGTCTAGAATGGGGATGGGGTCACTCTGGACCT. (SEQ ID NO:13)

These primers can be used, e.g., to amplify either the full lengthsequence or a probe of one to several hundred nucleotides, which is thenused to screen a library for full-length CNG2B. For example, Oligo 1(SEQ ID NO:4) can be used with Oligo 2 (SEQ ID NO:5) to produce a 657 bpband, and Oligo 9 (SEQ ID NO:12) and Oligo 10 (SEQ ID NO:13) can be usedto amplify the entire coding region. Further, in conjunction with otheroligos, Oligo 9 can be used with Oligos 2, 6, 7 or 8 (SEQ ID NOs:5, 9,10 or 11) to produce bands of approximately 1.7 Kb, 1.28 Kb, 170 bp, or90 bp, respectively. Similarly, Oligo 10 can be used with Oligos 1, 3,or 4 (SEQ ID NOs:4, 6 or 7) to produce fragments of approximately 715bp, 455 bp, or 240 bp, respectively. Only the nucleotides in bold typein oligos 9 and 10 are required for CNG2B amplification, and each of theabove product sizes are approximate.

Nucleic acids encoding CNG2B and other CNG2B family members can also beisolated from expression libraries using antibodies as probes. Suchpolyclonal or monoclonal antibodies can be raised using the sequence ofSEQ ID NO:1, or any immunogenic portion thereof.

CNG2B polymorphic variants, orthologs, and alleles that aresubstantially identical to the conserved region of CNG2B can be isolatedusing CNG2B nucleic acid probes and oligonucleotides under stringenthybridization conditions, by screening libraries. Alternatively,expression libraries can be used to clone CNG2B and CNG2B polymorphicvariants, orthologs, and alleles by detecting expressed homologsimmunologically with antisera or purified antibodies made against humanCNG2B or portions thereof, which also recognize and selectively bind tothe CNG2B homolog.

To make a cDNA library, one should choose a source that is rich in CNG2BmRNA, e.g., the central nervous system. The mRNA is then made into cDNAusing reverse transcriptase, ligated into a recombinant vector, andtransfected into a recombinant host for propagation, screening andcloning. Methods for making and screening cDNA libraries are well known(see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al.,supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grinstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating CNG2B nucleic acid and its orthologs,alleles, mutants, polymorphic variants, and conservatively modifiedvariants combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of human CNG2B directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify CNG2B homologs using the sequences provided herein.Restriction endonuclease sites can be incorporated into the primers.Polymerase chain reaction or other in vitro amplification methods mayalso be useful, for example, to clone nucleic acid sequences that codefor proteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of CNG2B encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of CNG2B can also be analyzed by techniques known in theart, e.g., reverse transcription and amplification of mRNA, isolation oftotal RNA or poly A⁺ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, high density polynucleotide arraytechnology and the like.

Synthetic oligonucleotides can be used to construct recombinant CNG2Bgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and non-sense (antisense)strands of the gene. These DNA fragments are then annealed, ligated andcloned. Alternatively, amplification techniques can be used with preciseprimers to amplify a specific subsequence of the CNG2B gene. Thespecific subsequence is then ligated into an expression vector.

The gene for CNG2B is typically cloned into intermediate vectors beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. These intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding CNG2B, one typically subclones the gene into an expressionvector that contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al., and Ausubel et al., supra. Bacterialexpression systems for expressing the CNG2B protein are available in,e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits forsuch expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the CNG2B encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding CNG2Band signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as MBP, GST, and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the CMV promoter, SV40early promoter, SV40 later promoter, metallothionein promoter, murinemammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrinpromoter, or other promoters shown effective for expression ineukaryotic cells.

Expression of proteins from eukaryotic vectors can be also be regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal. Inducible expression vectors are oftenchosen if expression of the protein of interest is detrimental toeukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with aCNG2B encoding sequence under the direction of the polyhedrin promoteror other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical—any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of CNG2Bprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,biolistics, liposomes, microinjection, plasma vectors, viral vectors andany of the other well known methods for introducing cloned genomic DNA,cDNA, synthetic DNA or other foreign genetic material into a host cell(see, e.g., Sambrook et al., supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingCNG2B.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofCNG2B, which is recovered from the culture using standard techniquesidentified below.

IV. Purification of CNG2B Polypeptides

Either naturally occurring or recombinant CNG2B can be purified for usein functional assays. Naturally occurring CNG2B monomers can bepurified, e.g., from human tissue such as the central nervous system orany other source of a CNG2B homolog. Recombinant CNG2B monomers can bepurified from any suitable expression system.

The CNG2B monomers may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant CNG2B monomersare being purified. For example, proteins having established molecularadhesion properties can be reversibly fused to the CNG2B monomers. Withthe appropriate ligand, the CNG2B monomers can be selectively adsorbedto a purification column and then freed from the column in a relativelypure form. The fused protein is then removed by enzymatic activity.Finally the CNG2B monomers could be purified using immunoaffinitycolumns.

A. Purification of CNG2B Monomers from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of the CNG2Bmonomers inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. Human CNG monomers areseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

Alternatively, it is possible to purify the CNG2B monomers from bacteriaperiplasm. After lysis of the bacteria, when the CNG2B monomers areexported into the periplasm of the bacteria, the periplasmic fraction ofthe bacteria can be isolated by cold osmotic shock in addition to othermethods known to skill in the art. To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

B. Standard Protein Separation Techniques for Purifying CNG2B Monomers

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of the CNG2B monomers (e.g., approximately 92 kD)can be used to isolate it from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column Chromatography

The CNG2B monomers can also be separated from other proteins on thebasis of size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

V. Immunological Detection of CNG2B Polypeptides

In addition to the detection of CNG2B genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect the CNG2B monomers of the invention. Immunoassays can be used toqualitatively or quantitatively analyze the CNG2B monomers. A generaloverview of the applicable technology can be found in Harlow & Lane,Antibodies: A Laboratory Manual (1988).

A. Antibodies to CNG2B Monomers

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with CNG2B monomers, or CNG2B monomers from particularspecies such as human CNG2B, are known to those of skill in the art(see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow &Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2ded. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of immunogens comprising portions of CNG2B monomers may be usedto produce antibodies specifically reactive with CNG2B monomers. Forexample, recombinant CNG2B monomers or an antigenic fragment thereof canbe isolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies may be generated,for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see, Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse, et al,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-CNG familyproteins and other CNG family proteins, using a competitive bindingimmunoassay. Specific polyclonal antisera and monoclonal antibodies willusually bind with a K_(d) of at least about 0.1 mM, more usually atleast about 1 M, preferably at least about 0.1 M or better, and mostpreferably, 0.01 M or better. Antibodies specific only for a particularCNG2B ortholog, such as human CNG2B, can also be made, by subtractingout other cross-reacting orthologs from a species such as a non-humanmammal.

Once the specific antibodies against a CNG2B are available, the CNG2Bcan be detected by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological Binding Assays

The CNG2B polypeptides of the invention can be detected and/orquantified using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology: Antibodies in Cell Biology, volume 37(Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds.,7^(th) ed. 1991). Immunological binding assays (or immunoassays)typically use an antibody that specifically binds to a protein orantigen of choice (in this case the CNG2B or an antigenic subsequencethereof). The antibody (e.g., anti-CNG2B) may be produced by any of anumber of means well known to those of skill in the art and as describedabove.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled CNG2B polypeptide ora labeled anti-CNG2B antibody. Alternatively, the labeling agent may bea third moiety, such a secondary antibody, which specifically binds tothe antibody/CNG2B complex (a secondary antibody is typically specificto antibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-competitive assay formats

Immunoassays for detecting the CNG2B in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-CNG2B subunit antibodies can bebound directly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture CNG2B present in the test sample.The CNG2B monomers are thus immobilized and then bound by a labelingagent, such as a second CNG2B antibody bearing a label. Alternatively,the second antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

Competitive Assay Formats

In competitive assays, the amount of the CNG2B present in the sample ismeasured indirectly by measuring the amount of known, added (exogenous)CNG2B displaced (competed away) from an anti-CNG2B antibody by theunknown CNG2B present in a sample. In one competitive assay, a knownamount of the CNG2B is added to a sample and the sample is thencontacted with an antibody that specifically binds to the CNG2B. Theamount of exogenous CNG2B bound to the antibody is inverselyproportional to the concentration of the CNG2B present in the sample. Ina particularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of CNG2B bound to the antibody may bedetermined either by measuring the amount of CNG2B present in aCNG2B/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of CNG2B may be detected byproviding a labeled CNG2B molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known CNG2B is immobilized on a solid substrate. A knownamount of anti-CNG2B antibody is added to the sample, and the sample isthen contacted with the immobilized CNG2B. The amount of anti-CNG2Bantibody bound to the known immobilized CNG2B is inversely proportionalto the amount of CNG2B present in the sample. Again, the amount ofimmobilized antibody may be detected by detecting either the immobilizedfraction of antibody or the fraction of the antibody that remains insolution. Detection may be direct where the antibody is labeled orindirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations for CNG2B. For example, a CNG2B proteinat least partially corresponding to an amino acid sequence of SEQ IDNO:1 or an immunogenic region thereof can be immobilized to a solidsupport. Other proteins such as other CNG family members are added tothe assay so as to compete for binding of the antisera to theimmobilized antigen. The ability of the added proteins to compete forbinding of the antisera to the immobilized protein is compared to theability of the CNG2B or immunogenic portion thereof to compete withitself. The percent crossreactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% crossreactivity with each of the added proteins listed above areselected and pooled. The cross-reacting antibodies are optionallyremoved from the pooled antisera by immunoabsorption with the addedconsidered proteins, e.g., distantly related homologs. Antibodies thatspecifically bind only to particular orthologs of CNG2B, such as humanCNG2B, can also be made using this methodology.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele, ortholog, or polymorphic variant ofCNG2B, to the immunogen protein. In order to make this comparison, thetwo proteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required to inhibit 50% of binding is less than 10 timesthe amount of the protein encoded by CNG2B that is required to inhibit50% of binding, then the second protein is said to specifically bind tothe polyclonal antibodies generated to the respective CNG2B immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of the CNG2B in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind CNG2B. The anti-CNG2B antibodies specificallybind to CNG2B on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-CNG2B antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,Amer. Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecule (e.g., streptavidin), which iseither inherently detectable or covalently bound to a signal system,such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize CNG2B, orsecondary antibodies that recognize anti-CNG2B antibodies.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see, U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally, simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies.

In this case, antigen-coated particles are agglutinated by samplescomprising the target antibodies. In this format, none of the componentsneed be labeled and the presence of the target antibody is detected bysimple visual inspection.

VI. Assays for Modulators of CNG2B

A. Assays

Introduction

Human CNG2B and CNG2B alleles, orthologs, and polymorphic variants aresubunits of cation channels. The activity of a cation channel comprisingCNG2B can be assessed using a variety of in vitro and in vivo assays,e.g., measuring current, measuring membrane potential, measuring ionflux, e.g., cations such as sodium or calcium, measuring ionconcentration, measuring second messengers and transcription levels,measuring ligand binding, and using, e.g., voltage-sensitive dyes, ionsensitive dyes such as cation (e.g., sodium or calcium) sensitive dyes,radioactive tracers, and patch-clamp electrophysiology.

In preferred embodiments, the activity of a CNG cation channel will bedetected by detecting cation, e.g., calcium or sodium, concentration orflux using an ion (e.g., calcium or sodium) specific dye, e.g., afluorescent dye. Any such dye, a large number of which are well known tothose of skill in the art, can be used. For example, any of a number offluorescent probes that show a spectral response upon binding Ca²⁺allowing the detection of changes in intracellular free Ca²⁺concentrations using fluorescence microscopy, flow cytometry orfluorescence spectroscopy, can be used.

Furthermore, such assays can be used to test for inhibitors andactivators of channels comprising CNG2B. Such modulators of a cationchannel are useful for treating various disorders involving cationchannels, e.g., neurological disorders, e.g., of the central nervoussystem. Such modulators are also useful for investigation of the channeldiversity provided by CNG family members and the regulation/modulationof cation channel activity provided by CNG family members such as CNG2B.

Modulators of the CNG cation channels are tested using biologicallyactive CNG2B, either recombinant or naturally occurring, preferablyhuman CNG2B. CNG2B can be isolated, co-expressed or expressed in a cell,or expressed in a membrane derived from a cell. In such assays, CNG2Bcan be expressed alone to form a homomultimeric cation channel, or incombination with other CNG proteins, including alpha and/or betasubunits, to form a heteromultimeric cation channel. Preferably, theCNG2B polypeptide that is a part of the cation channel used in the assaywill have the sequence displayed in SEQ ID NO:1 or a conservativelymodified variant thereof. Generally, the amino acid sequence identity ofthe polypeptide to SEQ ID NO:1 will be at least 60%, 65%, 70%, 75%, 80%,preferably 85%, or 90%, most preferably at least 95% or higher.

Modulation is tested using one of the in vitro or in vivo assaysdescribed herein Samples or assays that are treated with a potentialcation channel inhibitor or activator are compared to control sampleswithout the test compound, to examine the extent of modulation. Often,such assays are performed in the presence of a cyclic nucleotide, e.g.,cAMP or cGMP, and the ability of the test agent to modulate the effectof the cyclic nucleotide on the channel is detected.

Control samples (untreated with activators or inhibitors) are assigned arelative cation channel activity value of 100. Inhibition of channelscomprising a CNG2B polypeptide is achieved when the cation channelactivity value relative to the control is about 90%, preferably 50%,more preferably 25%. Activation of channels comprising a CNG2Bpolypeptide is achieved when the cation channel activity value relativeto the control is 110%, more preferably 150%, more preferable 200%higher. Compounds that increase the flux of ions will cause a detectableincrease in the ion current density by increasing the probability of achannel comprising a CNG2B polypeptide being open, by decreasing theprobability of it being closed, by increasing conductance through thechannel, and/or by allowing the passage of ions.

In Vitro Assays

Assays to identify compounds with cation channel modulating activity canbe performed in vitro, e.g., binding assays and biochemical assays.Purified recombinant or naturally occurring CNG2B protein, or a channelcomprising CNG2B protein, can be used in the in vitro methods of theinvention. In addition to purified CNG2B protein or channel comprisingthe same, the recombinant or naturally occurring CNG2B protein can bepart of a cellular lysate or a cell membrane. As described below, theassay can be either solid state or soluble. Preferably, the protein ormembrane is bound to a solid support, either covalently ornon-covalently. Often, the in vitro assays of the invention are ligandor toxin binding or ligand affinity assays, either non-competitive orcompetitive. Other in vitro assays include measuring changes inspectroscopic (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility propertiesfor the protein or channel. Cell membranes or lysates can also be usedto measure changes in polarization (i.e., electrical potential) of thecell or membrane expressing the cation channel comprising a CNG2Bpolypeptide, as described below.

In Vivo Cell—or Membrane Based Assays

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing the cation channel comprising a CNG2B polypeptide. Apreferred means to determine changes in cellular polarization is bymeasuring changes in current (thereby measuring changes in polarization)with voltage-clamp and patch-clamp techniques, e.g., the “cell-attached”mode, the “inside-out” mode, and the “whole cell” mode (see, e.g.,Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)). Whole cellcurrents are conveniently determined using standard methodology (see,e.g., Hamil et al., PFlugers. Archiv. 391:85 (1981). Other known assaysinclude fluorescence assays using ion sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J Membrane Biol. 88:67-75 (1988); Daniel etal., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J.Membrane Biology 137:59-70 (1994)).

Examples of such dyes useful for the detection of calcium include, butare not limited to, fura-2, bis-fura 2, indo-1, Quin-2, Quin-2 AM,Benzothiaza-1, Benzothiaza-2, indo-5F, Fura-FF, BTC, Mag-Fura-2,Mag-Fura-5, Mag-Indo-1, fluo-3, rhod-2, fura-4F, fura-5F, fura-6F,fluo-4, fluo-5F, fluo-5N, Oregon Green 488 BAPTA, Calcium Green,Calcein, Fura-C18, Calcium Green-C18, Calcium Orange, Calcium Crimson,Calcium Green-5N, Magnesium Green, Oregon Green 488 BAPTA-1, OregonGreen 488 BAPTA-2, X-rhod-1, Fura Red, Rhod-5F, Rhod-5N, X-Rhod-5N,Mag-Rhod-2, Mag-X-Rhod-1, Fluo-5N, Fluo-5F, Fluo-4FF, Mag-Fluo-4,Aequorin, dextran conjugates or any other derivatives of any of thesedyes, and others (see, e.g., the catalog or Internet site(www.probes.com) for Molecular Probes, Eugene, Oreg.; see, also,Nuccitelli, ed., Methods in Cell Biology, Volume 40: A Practical Guideto the Study of Calcium in Living Cells, Academic Press (1994); Lambert,ed., Calcium Signaling Protocols (Methods in Molecular Biology Volume114), Humana Press (1999); W. T. Mason, ed., Fluorescent and LuminescentProbes for Biological Activity. A Practical Guide to Technology forQuantitative Real-Time Analysis, Second Ed, Academic Press (1999)).Examples of sodium indicators include, but are not limited to, SBFI, andSodium Green (see, e.g., Molecular probes catalog or Internet site;Mason, supra).

Assays for compounds capable of inhibiting or increasing cation fluxthrough the channel proteins comprising a CNG2B polypeptide can beperformed by application of the compounds to a bath solution in contactwith and comprising cells having a channel of the present invention(see, e.g., Blatz et al., Nature 323:718-720 (1986); Park, J. Physiol.481:555-570 (1994)). Generally, the compounds to be tested are presentin the range from 1 μM to 100 mM.

The effects of the test compounds upon the function of the channels canbe measured by changes in the electrical currents or ionic flux or bythe consequences of changes in currents and flux. Changes in electricalcurrent or ionic flux are measured by either increases or decreases influx of ions such as sodium or calcium ions. The ions can be measured ina variety of standard ways. They can be measured directly byconcentration changes of the ions, e.g., changes in intracellularconcentrations, e.g., using any of the dyes listed supra, orradiolabeled ions, or indirectly by membrane potential. Consequences ofthe test compound on ion flux can be quite varied. Accordingly, anysuitable physiological change can be used to assess the influence of atest compound on the channels of this invention. The effects of a testcompound can be measured by a toxin binding assay. One can also measurea variety of effects such as transmitter release (e.g., dopamine),intracellular calcium changes, hormone release (e.g., insulin),transcriptional changes to both known and uncharacterized geneticmarkers (e.g., northern blots), cell volume changes (e.g., in red bloodcells), immunoresponses (e.g., T cell activation), changes in cellmetabolism such as cell growth or pH changes, and changes inintracellular second messengers such as cyclic nucleotides.

CNG2B orthologs, alleles, polymorphic variants, and conservativelymodified variants will generally confer substantially similar propertieson a channel comprising a CNG2B polypeptide, as described above. In apreferred embodiment, the cell placed in contact with a compound that issuspected to be a CNG2B homolog is assayed for increasing or decreasingion flux in a eukaryotic cell, e.g., an oocyte of Xenopus (e.g., Xenopuslaevis) or a mammalian cell such as a CHO or HeLa cell. Channels thatare affected by compounds in ways similar to CNG2B are consideredhomologs or orthologs of CNG2B.

Animal Models

Animal models also find use in screening for cation channel modulators.Transgenic animal technology, including gene knockout technology as aresult of homologous recombination with an appropriate gene targetingvector, or gene overexpression, will result in the absence or increasedexpression of the CNG2B protein. When desired, tissue-specificexpression or knockout of the CNG2B protein may be necessary. Transgenicanimals generated by such methods find use as animal models of abnormalion flux and are additionally useful in screening for modulators ofcation channels.

B. Modulators

The compounds tested as modulators of CNG channels comprising a CNG2Bsubunit can be any small organic molecule, or a biological entity, suchas a protein, peptide, sugar, nucleic acid, oligonucleotide, or lipid.Alternatively, modulators can be genetically altered versions of a CNG2Bsubunit. Typically, test compounds will be small chemical molecules andpeptides. Essentially any chemical compound can be used as a potentialmodulator or ligand in the assays of the invention, although most oftencompounds can be dissolved in aqueous or organic (especially DMSO-based)solutions are used. The assays are designed to screen large chemicallibraries by automating the assay steps and providing compounds from anyconvenient source to assays, which are typically run in parallel (e.g.,in microtiter formats on microtiter plates in robotic assays). It willbe appreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbarnates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see,e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.5,593,853), small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S.Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressinga CNG channel comprising a human CNG2B subunit is attached to a solidphase substrate. In the high throughput assays of the invention, it ispossible to screen up to several thousand different modulators orligands in a single day. In particular, each well of a microtiter platecan be used to run a separate assay against a selected potentialmodulator, or, if concentration or incubation time effects are to beobserved, every 5-10 wells can test a single modulator. Thus, a singlestandard microtiter plate can assay about 96 modulators. If 1536 wellplates are used, then a single plate can easily assay from about 100-about 1500 different compounds. It is possible to assay many plates perday; assay screens for up to about 6,000, 20,000, 50,000, or 100,000 ormore different compounds are possible using the integrated systems ofthe invention.

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provides soluble assays using cationchannels comprising a CNG2B polypeptide, a membrane comprising a CNG2Bcation channel, or a cell or tissue expressing cation channelscomprising a CNG2B polypeptide, either naturally occurring orrecombinant. In another embodiment, the invention provides solid phasebased in vitro assays in a high throughput format, where a CNG2B cationchannel is attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100- about 1500different compounds. It is possible to assay many plates per day; assayscreens for up to about 6,000, 20,000, 50,000, or more than 100,000different compounds are possible using the integrated systems of theinvention.

The channel of interest, or a cell or membrane comprising the channel ofinterest, can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage e.g., via a tag. Thetag can be any of a variety of components. In general, a molecule whichbinds the tag (a tag binder) is fixed to a solid support, and the taggedmolecule of interest is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherin family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly-gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39 (4):718-719(1993); and Kozal et al., Nature Medicine 2 (7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

VII. Computer Assisted Drug Design Using CNG2B

Yet another assay for compounds that modulate the activities of a CNG2Bchannel involves computer assisted drug design, in which a computersystem is used to generate a three-dimensional structure of CNG2B basedon the structural information encoded by the amino acid sequence. Theinput amino acid sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands or other cationchannel subunits. These regions are then used to identify ligands thatbind to the protein or region where CNG2B interacts with other cationchannel subunits.

The three-dimensional structural model of the protein is generated byentering channel protein amino acid sequences of at least 25, 50, 75,100, 150, or 200 amino acid residues or corresponding nucleic acidsequences encoding a CNG2B monomer into the computer system. The aminoacid sequence of each of the monomers is selected from the groupconsisting of SEQ ID NO:1, conservatively modified versions thereof, andimmunogenic portions thereof. The amino acid sequence represents theprimary sequence or subsequence of each of the proteins, which encodesthe structural information of the protein. At least 25, 50, 75, 100,150, or 200 residues of the amino acid sequence (or a nucleotidesequence encoding at least about 25, 50, 75, 100, 150, or 200 aminoacids) are entered into the computer system from computer keyboards,computer readable substrates that include, but are not limited to,electronic storage media (e.g., magnetic diskettes, tapes, cartridges,and chips), optical media (e.g., CD ROM), information distributed byinternet sites, and by RAM. The three-dimensional structural model ofthe channel protein is then generated by the interaction of the aminoacid sequence and the computer system, using software known to those ofskill in the art. The resulting three-dimensional computer model canthen be saved on a computer readable substrate.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the monomer and the heteromeric cation channel proteincomprising four monomers. The software looks at certain parametersencoded by the primary sequence to generate the structural model. Theseparameters are referred to as “energy terms,” or anisotropic terms andprimarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the CNG2B protein to identify ligands that bind to CNG2B.Binding affinity between the protein and ligands is determined usingenergy terms to determine which ligands have an enhanced probability ofbinding to the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of CNG2B genes. Suchmutations can be associated with disease states. Once the variants areidentified, diagnostic assays can be used to identify patients havingsuch mutated genes associated with disease states. Identification of themutated CNG2B genes involves receiving input of a first nucleic acid,e.g., SEQ ID NOS:2-3, or an amino acid sequence encoding CNG2B, e.g.,SEQ ID NO:1, and conservatively modified versions thereof. The sequenceis entered into the computer system as described above. The firstnucleic acid or amino acid sequence is then compared to a second nucleicacid or amino acid sequence that has substantial identity to the firstsequence. The second sequence is entered into the computer system in themanner described above. Once the first and second sequences arecompared, nucleotide or amino acid differences between the sequences areidentified. Such sequences can represent allelic differences in CNG2Bgenes, preferably human CNG2B genes, and mutations associated withdisease states. The first and second sequences described above can besaved on a computer readable substrate.

Nucleic acids encoding CNG2B monomers can be used with high densityoligonucleotide array technology (e.g., GeneChip™) to identify CNG2Bhomologs, orthologs, alleles, conservatively modified variants, andpolymorphic variants in this invention. In the case where the homologsbeing identified are linked to a known disease, they can be used withGeneChip™ as a diagnostic tool in detecting the disease in a biologicalsample, see, e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14:869-876 (1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al.,Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat. Biotechnol.14:1675-1680 (1996); Gingeras et al., Genome Res. 8:435-448 (1998);Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).

VIII. Cellular Transfection and Gene Therapy

The present invention provides the nucleic acids of CNG2B genes for thetransfection of cells in vitro and in vivo. These nucleic acids can beinserted into any of a number of well-known vectors for the transfectionof target cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The nucleic acid for CNG2B, under thecontrol of a promoter, then expresses a CNG2B monomer of the presentinvention, thereby mitigating the effects of absent, partialinactivation, or abnormal expression of the CNG2B gene. The compositionsare administered to a patient in an amount sufficient to elicit atherapeutic response in the patient. An amount adequate to accomplishthis is defined as “therapeutically effective dose or amount.” Such genetherapy procedures have been used to correct acquired and inheritedgenetic defects, cancer, and viral infection in a number of contexts.The ability to express artificial genes in humans facilitates theprevention and/or cure of many important human diseases, including manydiseases which are not amenable to treatment by other therapies (for areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6 (10):1149-1154 (1998); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51 (1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology (Doerfler & Böhm eds., 1995); and Yu et al.,Gene Therapy 1:13-26 (1994)).

Delivery of the gene or genetic material into the cell is the first stepin gene therapy treatment of disease. A large number of delivery methodsare well known to those of skill in the art. Preferably, the nucleicacids are administered for in vivo or ex vivo gene therapy uses.Non-viral vector delivery systems include DNA plasmids, naked nucleicacid, and nucleic acid complexed with a delivery vehicle such as aliposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell.

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described in,e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat.No. 4,897,355 and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Feigner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV),and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994)). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et al., Proc. Natl. Acad. Sci. U.S.A.92:9747-9751 (1995), reported that Moloney murine leukemia virus can bemodified to express human heregulin fused to gp70, and the recombinantvirus infects certain human breast cancer cells expressing humanepidermal growth factor receptor. This principle can be extended toother pairs of virus expressing a ligand fusion protein and target cellexpressing a receptor. For example, filamentous phage can be engineeredto display antibody fragments (e.g., FAB or Fv) having specific bindingaffinity for virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to nonviral vectors. Such vectors can be engineered tocontain specific uptake sequences thought to favor uptake by specifictarget cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a nucleicacid (gene or cDNA), and re-infused back into the subject organism(e.g., patient). Various cell types suitable for ex vivo transfectionare well known to those of skill in the art (see, e.g., Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. Suitablemethods of administering such nucleic acids are available and well knownto those of skill in the art, and, although more than one route can beused to administer a particular composition, a particular route canoften provide a more immediate and more effective reaction than anotherroute.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The nucleicacids are administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch nucleic acids are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

IX. Pharmaceutical Compositions and Administration

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., nucleic acid, protein,modulatory compounds or transduced cell), as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed., 1989). Administration can be in any convenient manner,e.g., by injection, oral administration, inhalation, or transdermalapplication.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles-comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of the CNG channels comprising a CNG2B subunit, thephysician evaluates circulating plasma levels of the vector, vectortoxicities, progression of the disease, and the production ofanti-vector antibodies. In general, the dose equivalent of a nakednucleic acid from a vector is from about 1 μg to 100 μg for a typical 70kilogram patient, and doses of vectors which include a retroviralparticle are calculated to yield an equivalent amount of therapeuticnucleic acid.

For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

X. Kits

Human CNG2B and its homologs are useful tools for examining expressionand regulation of cation channels. Human CNG2B-specific reagents thatspecifically hybridize to CNG2B nucleic acid, such as CNG2B probes andprimers, and CNG2B-specific reagents that specifically bind to the CNG2Bprotein, e.g., CNG2B antibodies, are used to examine expression andregulation.

Nucleic acid assays for the presence of CNG2B DNA and RNA in a sampleinclude numerous techniques are known to those skilled in the art, suchas Southern analysis, northern analysis, dot blots, RNase protection, S1analysis, amplification techniques such as PCR and LCR, and in situhybridization. In in situ hybridization, for example, the target nucleicacid is liberated from its cellular surroundings in such as to beavailable for hybridization within the cell while preserving thecellular morphology for subsequent interpretation and analysis. Thefollowing articles provide an overview of the art of in situhybridization: Singer et al., Biotechniques 4:230-250 (1986); Haase etal., Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic AcidHybridization: A Practical Approach (Hames et al., eds. 1987). Inaddition, CNG2B protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing recombinant CNG2Bmonomers) and a negative control.

The present invention also provides for kits for screening modulators ofthe cation channels of the invention. Such kits can be prepared fromreadily available materials and reagents. For example, such kits cancomprise any one or more of the following materials: CNG2B monomers,reaction tubes, and instructions for testing the activities of cationchannels containing CNG2B. A wide variety of kits and components can beprepared according to the present invention, depending upon the intendeduser of the kit and the particular needs of the user. For example, thekit can be tailored for in vitro or in vivo assays for measuring theactivity of a cation channel comprising a CNG2B monomer.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

A. Identification of Human CNG2B

Multiple exons of human CNG2B were identified from public genomic data(accession numbers AC022762 and AC021935) using TBLASTN searches withcyclic nucleotide-gated channel protein sequences. The 5′ and 3′ exonsof the CNG2B coding sequence could not be identified in these searches.Oligonucleotides based on the AC022762 and AC021935 sequences weredesigned to clone a full-length CNG2B cDNA.

An approximately 657 bp band from the CNG2B gene was amplified fromfirst strand cDNA prepared from the human brain, demonstratingexpression in the central nervous system. The oligos used to amplifythis band were 5′-(1) GCAGATCTTCCAGAACTGTAAGGCCA (SEQ ID NO:14) (sense)and 5′-(2) CCTGCCCTCTTCATCTTTGGAAGTTC (SEQ ID NO:5) (antisense). Thecomplete 3′ end of CNG2B was amplified by standard 3′ RACE PCRtechniques from human brain cDNA in two successive rounds. In the firstround the gene specific primer used was 5′-(3)GCCAACATCAAGAGCCTAGGTTATTC (SEQ ID NO:6) (sense). This reaction was thenreamplified with a nested gene specific oligo 5′-(4)GGATGATCTACAGACCAAGTTTGCTCG (SEQ ID NO:7) (sense) to produce a fragmentof approximately 765 bp in length that, when sequenced, was found toinclude the complete 3′ end of the human CNG2B mRNA. The sequence ofthis fragment overlapped with the original 657 bp CNG2B fragment toprovide contiguous sequence. Most of the 5′ end of the CNG2B codingsequence was amplified from human brain cDNA using a degenerate sensestrand oligo based on the N-terminal amino acid sequence of rat OCNC2protein (5′-(5) ATGAGCCAGGACGGNAARGTNAARAC (SEQ ID NO:15)) and anantisense primer specific to human CNG2B (5′-(6)GTTGATGATGCTGATCTCCCCAAAG (SEQ ID NO:9)). This reaction produced afragment of approximately 1.2 Kb with a sequence highly homologous torat OCNC2. Two rounds of standard 5′ RACE PCR were then used to completethe 5′ coding sequence of human CNG2B and to identify the initiatormethionine codon. The CNG2B-specific oligo 5′-(7)GGATGATGAGGTTATACATGACTGGG (SEQ ID NO:10) (antisense) was used in thefirst round of RACE PCR. This reaction was reamplified using the nestedCNG2B specific oligo 5′-(8) AGGCTAGCAACTTCCTGGCCTTGGAT (SEQ ID NO:11)(antisense). An approximately 410 bp fragment containing the complete 5′end of CNG2B including the start codon was isolated. This fragmentoverlapped the 1.2 Kb PCR fragment described above. The entirecontiguous coding region of the CNG2B mRNA was determined by assemblingthese two fragments with the original 657 bp internal fragment and the765 bp 3′ RACE product.

The entire coding region of human CNG2B was then isolated in a singlefragment using oligonucleotides overlapping the CNG2B coding sequenceends. The oligonucleotides used were 5′-(9)GCGAAAGCTTCCACCATGAGCCAGGACACCAAAGTG (SEQ ID NO:12) (sense) and 5′-(10)CATGTCTAGAATGGGGATGGGGTCACTCTGGACCT (SEQ ID NO:13) (antisense). Thefirst oligonucleotide includes the initiator methionine and the first 21coding nucleotides of the CNG2B gene. Upstream are a HindIII restrictionenzyme site for subcloning into plasmid vectors and a Kozak consensussequence to boost translation. All nucleotides corresponding to CNG2Bare in bold type. The second oligonucleotide is from the 3′ sequence ofCNG2B and includes an XbaI restriction enzyme site for subcloning. Allnucleotides in bold correspond to the 3′ end sequence of CNG2B. The stopcodon is underlined. It is important to note that only the nucleotidesthat are in bold type from the two oligos above are necessary foramplification of CNG2B. The preferred template for the amplification isfirst strand cDNA from the human brain. The amplification conditionsused were as follows: 24 cycles of 95° C. for 15 seconds, 72-60° C. for15 seconds (temperature was dropped 0.5° C. each successive cycle), 72°C. for 3 minutes, followed by 20 cycles of 95° C. for 15 seconds, 60° C.for 15 seconds, and 72° C. for 3 minutes. An approximately 1.73 Kb bandcorresponding to the entire coding region of CNG2B was obtained andconfirmed by sequencing.

The predicted molecular weight of the human CNG2B protein is about 66Kd, with a range of approximately 60 Kd-80 Kd, preferably about 61-71Kd.

B. Comparison of Human CNG2B with Other CNG Genes

An alignment of the deduced amino acid sequence of CNG2B to rat OCNC2(Bradley, et al., Proc. Natl. Acad. Sci. U.S.A. 91, 8890-8894, 1994) isshown in FIG. 1. The amino acid sequences of human CNG2B and rat OCNC2are 93% identical, indicating that they are likely to be orthologousgenes. Additional evidence supporting this idea is that human CNG2B ismuch more homologous to rat OCNC2 than any of the other cloned CNGchannels. Most of the differences between the two amino acid sequencesare clustered at the amino and carboxy termini. Human CNG2B and ratOCNC2 are less than 90% identical on the nucleic acid level.

The human CNG2B gene appears to be orthologous to the rat OCNC2 gene,suggesting that it serves a similar functional role. In support of thisidea is our evidence for expression of human CNG2B in the brain, wherethere is widespread expression of rat OCNC2 (Kingston, et al., Synapse32:1-12 (1999). The rat OCNC2 gene was originally classified as a CNGbeta subunit because it is functionally insensitive to cyclicnucleotides when expressed as a homomultimer (Bradley, et al., Proc.Nat. Acad. Sci. 91:8890-8894 (1994). Instead, it was shown to formfunctional heteromultimeric channels with the rat OCNC1 alpha subunitwhich participates in olfactory transduction (Bradley, et al., Proc.Nat. Acad. Sci. 91:8890-8894 (1994). This alpha and betaheteromultimeric channel showed increased sensitivity to cAMP closelyresembling the native CNG olfactory channel (Linman, et al., Neuron13:611-621 (1994). However, other studies have shown that functionalhomomeric rat OCNC2 channels may exist in the brain, and that they arenitric oxide-sensitive (Broillet, et al., Neuron 18:95.1-958 (1997)).This finding, combined with the widespread distribution of rat OCNC2 inthe brain and its high permeability to Ca²⁺, suggest that these channelsmay play a role in neuronal signaling and synaptic plasticity (Bradley,et al., JNC 17:1993-2005 (1997). The ability of rat OCNC2 to formfunctional homomultimeric channels is consistent with the fact that itshares greater homology with CNG alpha subunits than with CNG betasubunits. Rat OCNC2 and human CNG2B are thus likely to be functionallysignificant both as heteromultimers and as homomultimers.

1-10. (canceled)
 11. An isolated polypeptide comprising a subunit of acation channel, the polypeptide: (i) forming, with at least one cyclicnucleotide gated cation channel (CNG) alpha subunit, a cation channelhaving the characteristic of cyclic nucleotide-gating; and (ii)comprising an amino acid sequence of SEQ ID NO:1.
 12. (canceled)
 13. Thepolypeptide of claim 11, wherein the polypeptide has a molecular weightof between about 61 kD to about 71 kD.
 14. (canceled)
 15. Thepolypeptide of claim 11, wherein the polypeptide consists of an aminoacid sequence of SEQ ID NO:1.
 16. The polypeptide of claim 11, whereinthe polypeptide comprises an alpha subunit of a heteromeric cyclicnucleotide-gated cation channel. 17-40. (canceled)